MicroRNA Gene Expression Mapping in Chicken Embryos

描述（由申请人提供）：本竞争性补充申请的目的是通过全囊原位杂交绘制发育第8天鸡胚中所有microrna的表达图谱，并优化使用短rna探针检测体内mrna的技术。这将扩大目前资助的GEISHA（鸡原位杂交分析）项目（HD044767），该项目的目标是绘制鸡胚胎发育第4天所有编码mrna的蛋白质的表达，将这些信息与鸡基因组浏览器联系起来，并通过免费访问的用户界面（http://geisha.arizona.edu）呈现信息。MicroRNAs通过结合靶mrna的3' utr序列进行转录后调控基因表达，是一种新的生物学调控机制，在动物生长发育和疾病中具有重要作用。绘制microRNA表达模式目前不是GESIHA项目的目标，因为用于mrna原位杂交检测的标准反义RNA探针不能检测microRNA，其成熟形式只有21-23nt。虽然标准的DNA寡核苷酸也没有被证明是有用的原位杂交探针，但一类新的DNA类似物被称为锁定核酸（LNAs），最近被开发出来，具有显着改善的杂交特性，热稳定性和特异性。初步研究表明，21-23核苷酸LNA探针可以很容易地检测体内的microrna，并且通过协议修改也可以检测mrna。对于这个补充，我们将使用这项技术来实现两个具体目标；1)利用全载和切片原位杂交方法，绘制鸡胚发育第8天所有microrna的表达图谱。2)优化利用短rna探针原位杂交检测编码mrna的蛋白。利用短rna在体内检测mrna的能力将允许直接根据序列信息设计探针，从而消除了生产探针所需的cDNA模板。这项新技术可以从根本上改变原位杂交分析的方式。

DESCRIPTION (provided by applicant): The purpose of this competing supplemental application is to map by whole mount in situ hybridization the expression of all microRNAs in the chicken embryo through day 8 of development, and to optimize technologies for detecting mRNAs in vivo using short LNA probes. This will expand the presently funded GEISHA (gallus est in situ hybridization analysis) project (HD044767), the aims of which are to map expression of all protein encoding mRNAs through day 4 of chick embryo development, to link this information to the chicken genome browsers and to present information through a freely accessible user interface (http://geisha.arizona.edu). MicroRNAs posttranscriptionally regulate gene expression by binding to sequences in the 3'UTRs of target mRNAs, and represent a new mechanism of biological regulation with important functions in animal growth, development and disease. Mapping microRNA expression patterns is not presently an objective of the GESIHA project because standard antisense RNA probes used for in situ hybridization detection of mRNAs cannot detect microRNAs, the mature forms of which are only 21-23nt. Although standard DNA oligonucleotides have also not proven useful as in situ hybridization probes, a new class of DNA analogues called locked nucleic acids (LNAs) have recently been developed that exhibit significantly improved hybridization characteristics, thermal stability and specificity. Preliminary studies demonstrate that 21-23 nucleotide LNA probes can readily detect microRNAs in vivo, and through protocol modifications can also detect mRNAs. For this supplement we will use this technology to pursue two specific aims; 1) map the expression of all chicken microRNAs through day 8 of embryo development, using whole mount and section in situ hybridization approaches. 2) Optimize the use of short LNA probes for in situ hybridization detection of protein encoding mRNAs. The ability to use short LNAs to detect mRNAs in vivo will allow probes to be designed directly from sequence information, eliminating the need for a cDNA template for probe production. This new technology could fundamentally change the way in which in situ hybridization analyses are performed.